Yukari Mimura

Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells

Abstract Bone morphogenetic proteins (BMPs) play critical roles in folliculogenesis by modulating the actions of follicle-stimulating hormone (FSH) in the ovary. However, the effects of FSH on the BMP system remain unknown. Here, we have investigated the effects of FSH on BMP signaling using the human granulosa-like tumor cell line KGN. KGN cells express BMP type I and type II receptors and the BMP signaling molecules SMADs. FSH administration upregulated BMP type IA (BMPR1A) and IB (BMPR1B) receptors, activin type II receptor (ACVR2), and BMP type II receptor (BMPR2). FSH also augmented SMAD1 and SMAD5 expression, and conversely, FSH suppressed the expression of the inhibitory SMADs, SMAD6 and SMAD7. Bioassays revealed that FSH enhances BMP-induced SMAD1/5/8 phosphorylation and cellular DNA synthesis induced by BMP6 and BMP7. Since overexpression of BMPR1A and BMPR1B, but not SMADs, significantly enhanced the BMP responses, these type I receptors were revealed to be limiting factors for BMP signaling in KGN cells. BMPs significantly suppressed progesterone synthesis induced by forskolin and dibutyryl-cAMP (BtcAMP) but had no effect on estradiol induced by the same factors. KGN cAMP levels induced by forskolin were not altered by BMPs, suggesting that BMPs regulate steroidogenesis at a level downstream of cAMP synthesis in KGN cells. In this regard, BMPs specifically reduced the STAR transcription, whereas the levels of CYP11A, HSD3B2, and CYP19 stimulated by forskolin as well as BtcAMP were not altered. Collectively, the two major factors, FSH-cAMP pathway and BMP system, are reciprocally and functionally linked. Given that BMPs downregulate FSH receptors in KGN cells, this interaction may contribute to fine-tuning of the mutual sensitivity toward BMP ligands and FSH.

Effects of chronic inhibition of ACE and AT1 receptors on glomerular injury in Dahl salt-sensitive rats

To elucidate the contribution of the renin-angiontensin system (RAS) to glomerular injury in salt-sensitive hypertension, we investigated the chronic effects of the angiotensin I-converting enzyme inhibitor cilazapril and the angiotensin II type 1-receptor antagonist (AT1a) TCV-116 in Dahl-Iwai rats. Dahl salt-sensitive (S) rats receiving 8% salt diet for 6 wk were simultaneously treated with cilazapril ( n = 6), TCV-116 ( n = 6), or saline ( n = 14). The 8% salt diet markedly increased systolic blood pressure (SBP), urinary protein, and N-acetyl-β-glucosaminidase (NAG) excretion compared with 0.3% salt-treated S ( n = 6) or salt-resistant ( n = 6) rats. Although neither cilazapril nor TCV-116 reduced the elevated SBP, TCV-116 significantly lowered urinary protein and NAG excretion. Histologically, 8% salt treatment in S rats induced progressive sclerotic and proliferative glomerular changes, which were ameliorated by both drugs. TCV-116 increased the glomerular diameter. Immunofluorescence demonstrated the increased level of type III collagen in the mesangium of 8% salt-treated S rats, which was completely reversed by TCV-116. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that 8% salt treatment significantly increased the levels of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B-chain and that TCV-116 significantly reduced the levels of PCNA and transforming growth factor-β1 (TGF-β1). Thus, although the chronic RAS-inhibition in salt-sensitive hypertension exerted a histologically renoprotective effect by both ways without lowering blood pressure, the RAS inhibition due to AT1a had more beneficial advantages of reducing proteinuria and attenuating the levels of glomerular TGF-β1 and extracellular matrix.To elucidate the contribution of the renin-angiontensin system (RAS) to glomerular injury in salt-sensitive hypertension, we investigated the chronic effects of the angiotensin I-converting enzyme inhibitor cilazapril and the angiotensin II type 1-receptor antagonist (AT1a) TCV-116 in Dahl-Iwai rats. Dahl salt-sensitive (S) rats receiving 8% salt diet for 6 wk were simultaneously treated with cilazapril (n = 6), TCV-116 (n = 6), or saline (n = 14). The 8% salt diet markedly increased systolic blood pressure (SBP), urinary protein, and N-acetyl-beta-glucosaminidase (NAG) excretion compared with 0.3% salt-treated S (n = 6) or salt-resistant (n = 6) rats. Although neither cilazapril nor TCV-116 reduced the elevated SBP, TCV-116 significantly lowered urinary protein and NAG excretion. Histologically, 8% salt treatment in S rats induced progressive sclerotic and proliferative glomerular changes, which were ameliorated by both drugs. TCV-116 increased the glomerular diameter. Immunofluorescence demonstrated the increased level of type III collagen in the mesangium of 8% salt-treated S rats, which was completely reversed by TCV-116. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that 8% salt treatment significantly increased the levels of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B-chain and that TCV-116 significantly reduced the levels of PCNA and transforming growth factor-beta1 (TGF-beta1). Thus, although the chronic RAS-inhibition in salt-sensitive hypertension exerted a histologically renoprotective effect by both ways without lowering blood pressure, the RAS inhibition due to AT1a had more beneficial advantages of reducing proteinuria and attenuating the levels of glomerular TGF-beta1 and extracellular matrix.

Effects of peroxisome proliferator-activated receptor activation on gonadotropin transcription and cell mitosis induced by bone morphogenetic proteins in mouse gonadotrope LβT2 cells

Involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma ) activation and bone morphogenetic protein (BMP) signaling in regulating cell proliferation and hormonal production of pituitary tumors has been reported, although the underlying mechanism remains poorly understood. Here, we investigated regulatory roles of PPARalpha and PPARgamma in gonadotropin transcription and cell mitosis modulated by pituitary activin/BMP systems using a mouse gonadotropinoma cell line Lbeta T2, which expresses activin/BMP receptors, transcription factor Smads, PPARalpha , and PPARgamma . In Lbeta T2 cells, BMP signaling shown by Smad1/5/8 phosphorylation and Id-1 transcription was readily activated by BMPs. A PPARgamma agonist, pioglitazone significantly reduced BMP-induced DNA synthesis by Lbeta T2; whereas the PPARalpha agonist, fenofibric acid, did not. In accordance with the effects on cell mitosis, pioglitazone but not fenofibric acid significantly decreased BMP-induced Id-1-Luc activation. Neither fenofibric acid nor pioglitazone affected activin signaling detected by (CAGA)9-Luc activity. Both PPARalpha and PPARgamma ligands directly suppressed transcriptional activities of FSHbeta , LHbeta , and GnRHR. Activation of PPARalpha and PPARgamma increased mRNA levels of follistatin, but did not affect the expression of follistatin-related gene. Thus, PPAR agonists not only directly suppress gonadotropin transcription and BMP signaling, but also inhibit the biological actions of activins which facilitate gonadotropin transcription through upregulating follistatin expression. In addition, pioglitazone increased BMP ligands mRNA, but decreased activin-beta B mRNA in Lbeta T2 cells. Collectively, PPAR activation differentially regulates gonadotrope cell proliferation and gonadotropin transcription in a ligand-dependent manner.

Enhancement of aldosterone-induced catecholamine production by bone morphogenetic protein-4 through activating Rho and SAPK/JNK pathway in adrenomedullar cells

Here we investigated the effects of mineralocorticoid in the regulation of catecholamine biosynthesis using rat pheochromocytoma PC12 cells. Expression of mineralocorticoid receptor (MR) was confirmed in undifferentiated PC12 cells. Aldosterone stimulated dopamine production by PC12 cells without any increase in cAMP activity. Aldosterone-induced dopamine accumulation was enhanced in accordance with the increase in the rate-limiting enzyme tyrosine hydroxylase (TH). Blocking MR with eplerenone suppressed aldosterone-induced increases of TH mRNA and dopamine production. A glucocorticoid receptor (GR) antagonist, RU-486, attenuated dexamethasone- but not aldosterone-induced TH expression. Cycloheximide reduced both aldosterone- and dexamethasone-induced TH mRNA. A SAPK/JNK inhibitor, SP600125, suppressed aldosterone-induced TH mRNA expression; however, the aldosterone-induced TH expression was not affected by inhibition of ERK1/2, p38-MAPK, Rho-kinase, PI 3-kinase, and PKC. It was of note that cotreatment with eplerenone and SP600125 restored aldosterone-induced TH mRNA expression to basal levels. To investigate the involvement of bone morphogenetic protein (BMP) actions in aldosterone-induced catecholamine production, we examined the effects of BMP-4 and BMP-7, which are expressed in the adrenal medulla, on catecholamine biosynthesis. BMP-4 preferentially enhanced aldosterone-induced TH mRNA and dopamine production, although BMP-4 alone did not affect TH expression. The BMP-4 enhancement of aldosterone-induced TH expression was not observed in cells treated with eplerenone. BMP-4 did not affect MR expression of PC12 cells; however, it did enhance aldosterone-induced SAPK/JNK phosphorylation. Inhibition of SAPK/JNK or Rho suppressed BMP-4 enhancement of aldosterone-induced TH expression. Collectively, our findings demonstrate that aldosterone stimulates catecholamine biosynthesis in adrenomedullar cells via MR through genomic action and partly through nongenomic action by Rho-SAPK/JNK signaling, the latter of which is facilitated by BMP-4. A functional link between MR actions and endogenous BMP may be involved in the catecholamine production.

The role of nitric oxide and the renin-angiotensin system in salt-restricted Dahl rats

To elucidate the role of nitric oxide (NO) and renin-angiotensin system (RAS) in the development of salt-sensitive hypertension, we investigated the pressor responses and renal histologic changes after long-term inhibition of endogenous NO synthesis in Dahl-Iwai salt-sensitive (DS) and salt-resistant (DR) rats under salt-re-stricted conditions that exaggerate RAS activation. Male DS and DR rats (6 weeks old) were fed with a low-salt (0.3%) diet for 5 weeks. NG-nitro-L-arginine (L-NA; dissolved in 60 mg/L deionized water), an arginine analogue acting as a NO-inhibitor, was also administered for 5 weeks. L-NA administration induced a gradual increase in systolic blood pressure (SBP) in both strains, and the pressor response in DS rats was apparently more enhanced relative to that in DR rats. Urinary nitrate plus nitrite (u-NOx) excretion was decreased by L-NA, with a significant negative correlation between SBP and u-NOx excretion in DS rats but not in DR rats. Plasma renin activity and urinary aldosterone level were significantly increased in L-NA-treated DS rats on week 5. Marked histologic changes with glomerular sclerosis and increased proteinuria and urinary N-acetyl-beta-glucosaminidase excretion were found in L-NA-treated DS rats but not DR rats. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that angiotensin II type 1 receptor (AT1R) mRNA level was significantly lower in DS rats than in DR rats at week 2, and that L-NA administration significantly reduced glomerular AT1R level of DS rats at week 5, possibly because of downregulation. Our results showed that, even under sodium restriction, the pressor response and renal injury induced by chronic NO inhibition were markedly more enhanced in DS rats than in DR rats, which indicates that depletion of NO participates in both the development of hypertension and glomerular injury in DS rats through a potential activation of RAS irrespective of sodium loading. These data suggest that endogenous NO is an essential determinant of salt-sensitive hypertension in DS rats.

In vitro Macro-and Microautoradiographic Localization of V1 and V2 Receptors in the Rat Kidney Using OPC-21268 and OPC-31260

To elucidate the precise localization of vasopressin (VP) V1 and V2 receptors in the kidney, we utilized in vitro macroautoradiography (macro-ARG) and microautoradiography (micro-ARG) of these receptors in Wistar rat kidneys. This was done by using OPC-21268 and OPC-31260, two newly developed selective V1 (OPC-21268) and V2 (OPC-31260) receptor antagonists. For macro-ARG, 10-microm kidney sections were incubated with Tris-HCl buffer containing [3H]-VP with or without unlabeled ligand (VP, OPC-21268, or OPC-31260) at 20 degrees C for 40 min. These sections were then loaded into X-ray cassettes with Hyperfilm-[3H] and exposed in the dark for 2 months. The autoradiograms were quantitatively analyzed by using the research analysis system RAS 1,000; the V1 and V2 receptors were quantitated by subtracting the nonspecific binding (incubated with OPC-21268 and OPC-31260, respectively) from the total binding. To assess a more precise localization of the V1 and V2 receptors, we also investigated the micro-ARG of the renal V1 and V2 receptors by dipping the kidney section slides used for macro-ARG into a photographic emulsion and observing the receptors under light microscopy. [3H]-VP binding to the rat kidney was completely displaced by unlabeled excess VP, but not by unlabeled angiotensin II, indicating that [3H]-VP binding was specific for VP receptors. Computerized quantification showed that V2 receptors, visualized by OPC-31260, were the predominant type of VP receptor in the kidney. Conversely, V1 receptors, visualized by OPC-21268, were fewer in number. V1 receptors were partly localized to the glomerulus, cortical vessels, interstitial cells, and the medullary vessels. The V2 receptors localized to the collecting ducts and medullary tubules. Our findings indicated that renal V1 and V2 receptors can be detected by in vitro macro- and micro-ARG by using OPC-21268 and OPC-31260.

Differential effect of chronic inhibition of calcium channel and angiotensin II type 1-receptor on aldosterone synthesis in spontaneously hypertensive rats.

We have investigated the in vivo effect of chronic blockade of Ca(2+)-channels and angiotensin II type 1 (AT(1))-receptors on aldosterone (Aldo)-synthesis in the adrenal glands of spontaneously hypertensive rats (SHR). Male SHR were administered Ca(2+)-antagonist, amlodipine (10 mg/kg per day) or AT(1)-receptor-antagonist, TCV-116 (1 mg/kg per day) from 7 until 11 weeks of age. Systolic blood pressure (SBP) and heart rate (HR) were significantly higher in SHR than Wistar-Kyoto (WKY) rats. Both treatments resulted in equivalent and significant reduction in SBP in SHR. Aldo-secretion in SHR, which was significantly higher than in WKY rats, was profoundly suppressed by TCV-116 compared with amlodipine. Both treatments resulted in thickening of the zona glomerulosa, which immunohistochemically contains Aldo, at the end of therapy. Competitive reverse transcription-polymerase chain reaction (RT-PCR) showed that CYP11A (P450scc) mRNA regulating the first step of Aldo-synthesis was significantly reduced from week 9 of age by amlodipine, and that CYP11B2 (P450aldo) mRNA regulating the last step of Aldo-synthesis was potently suppressed from 9 weeks of age by TCV-116. Our results indicate that chronic treatment with different antihypertensive agents directly modulates adrenocortical aldosterone synthesis in SHR in vivo via different mechanisms.

Relationship between adrenomedullin and vasopressin-aquaporin system under general anesthesia.

Aim: The roles of adrenomedullin (AM) in body fluid balance under general anesthesia were investigated. Methods: Time course changes in plasma osmolality, AM, arginine vasopressin (AVP), and urinary aquaporin 2 (AQP2) in 17 patients undergoing abdominal surgery under general anesthesia were examined. Results: Increases in plasma AM levels were observed in parallel with increases in the levels of urinary AQP2/creatinine (Cr) before induction and 90 and 180 min after initiation of anesthesia. Significant correlations between plasma AM and urinary AQP2/Cr (r = 0.62, p < 0.0001) as well as urinary AVP/Cr and AQP2/Cr (r = 0.60, p < 0.0001) were uncovered. Multivariate stepwise analysis identified plasma AM as the critical independent factor affecting urinary AQP2/Cr level. Conclusion: A novel correlation of AM and AQP2 which overlays an AVP-AQP2 system may play a key role in fluid homeostasis during general anesthesia.

Testosterone modulates serum leptin concentrations in a male patient with hypothalamic hypogonadism

Serial measurements of body mass index (BMI), serum concentrations of testosterone (T), estradiol (E) and leptin (L) were performed before and after gonadotropin (Gn) therapy in an 18-year-old male subject (BMI 25.4 kg/m2) with idiopathic hypothalamic hypogonadism (IHH). We also measured the BMI and serum concentrations of L in 99 age-matched healthy subjects. Serum L correlated significantly with BMI in control subjects (r=0.84, p<0.0001). Baseline serum concentrations of L in our case were markedly high and both T and E were very low, but Gn therapy resulted in a gradual decrease in L and improvement in T and E, finally reaching the control levels of BMI-matched subjects. Our results demonstrate that T is a powerful negative modulator of serum L independent of BMI in conditions associated with low T levels, such as IHH.

Chronic treatment with amlodipine modulates adrenocortical angiotensin II receptors in spontaneously hypertensive rats.

We investigated the effects of long-term treatment with calcium-antagonist, amlodipine, on angiotensin II receptors in the adrenal cortex of spontaneously hypertensive rats (SHR). Seven-week-old male SHR were treated with oral amlodipine (10 mg/kg/day) or vehicle (saline) for four weeks. Age-matched normotensive Wistar-Kyoto (WKY) rats were treated with the vehicle similar to control SHR. Systolic blood pressure (SBP) showed time-dependent increase in SHR but not in WKY rats, while amlodipine treatment significantly reduced the high SBP in SHR. Plasma renin activity was serially increased in SHR, which was further enhanced by amlodipine treatment. But the plasma aldosterone level which was increased in SHR was not changed by amlodipine. Competitive reverse transcriptase-polymerase chain reaction showed that the level of adrenocortical angiotensin II type 1 receptor (AT1R) mRNA progressively decreased in vehicle-treated SHR compared to WKY rats and that 4-week course of amlodipine treatment significantly increased AT1R mRNA in SHR to levels comparable to those in WKY rats. Amlodipine treatment reduced the level of adrenocortical angiotensin II type 2 receptor (AT2R) mRNA in SHR from 8 weeks of age. Thus, chronic amlodipine treatment differently modulates both adrenocortical AT1R and AT2R in SHR in a possibly direct manner.